Techniques based on nuclear magnetic resonance (NMR) for imaging and chemical analyses of in vivo, or otherwise intact, biological systems are rapidly emerging and finding diverse applications within a wide range of fields. Very recently, several NMR-based techniques have been developed for the zebrafish as a model animal system. In the current study, the novel application of high-resolution magic angle spinning (HR-MAS) NMR is presented as a means of metabolic profiling of intact zebrafish embryos. Toward investigating the utility of HR-MAS NMR as a toxicological tool, these studies specifically examined metabolic changes of embryos exposed to polymethoxy-1-alkenes (PMAs)-a recently identified family of teratogenic compounds from freshwater algae-as emerging environmental contaminants. One-dimensional and two-dimensional HR-MAS NMR analyses were able to effectively identify and quantify diverse metabolites in early-stage (£36 h postfertilization) embryos. Subsequent comparison of the metabolic profiles between PMA-exposed and control embryos identified several statistically significant metabolic changes associated with subacute exposure to the teratogen, including (1) elevated inositol as a recognized component of signaling pathways involved in embryo development; (2) increases in several metabolites, including inositol, phosphoryl choline, fatty acids, and cholesterol, which are associated with lipid composition of cell membranes; (3) concomitant increase in glucose and decrease in lactate; and (4) decreases in several biochemically related metabolites associated with central nervous system development and function, including c-aminobutyric acid, glycine, glutamate, and glutamine. A potentially unifying model/hypothesis of PMA teratogenicity based on the data is presented. These findings, taken together, demonstrate that HR-MAS NMR is a promising tool for metabolic profiling in the zebrafish embryo, including toxicological applications.
Diets must satisfy the everyday metabolic requirements of organisms and can also serve as medicines to combat disease. Currently, the medicinal role of diets is much better understood in terrestrial than in aquatic ecosystems. This is surprising because phytoplankton species synthesize secondary metabolites with known antimicrobial properties. Here, we investigated the medicinal properties of phytoplankton (including toxin-producing cyanobacteria) against parasites of the dominant freshwater herbivore, Daphnia. We fed Daphnia dentifera on green algae and toxic cyanobacteria diets known to vary in their nutritional quality and toxin production, and an additional diet of Microcystis with added pure microcystin-LR. We then exposed Daphnia to fungal and bacterial parasites. Anabaena, Microcystis and Chlorella diets prevented infection of Daphnia by the fungal parasite Metschnikowia, while Nodularia toxins increased offspring production by infected hosts. In contrast to their medicinal effects against Metschnikowia, toxic phytoplankton generally decreased the fitness of Daphnia infected with the bacterial parasite, Pasteuria. We also measured the amount of toxin produced by phytoplankton over time. Concentrations of anatoxin-a produced by Anabaena increased in the presence of Metschnikowia, suggesting parasite-induced toxin production. Our research illustrates that phytoplankton can serve as toxins or medicines for their consumers, depending upon the identity of their parasites.
Cylindrospermopsis raciborskii is among the most commonly recognized toxigenic cyanobacteria associated with harmful algal blooms (HAB) in freshwater systems, and specifically associated with multiple water-soluble toxins. Lipophilic metabolites from C. raciborskii, however, were previously shown to exert teratogenicity (i.e. inhibition of vertebrate development) in the zebrafish (Danio rerio) embryo model, specifically suggesting the presence of additional bioactive compounds unrelated to the currently known toxins. In the present study, a series of known teratogenic polymethoxy-1-alkenes (PMA) were identified, purified and chemically characterized from an otherwise well-characterized strain of toxigenic C. raciborskii. Although PMA have been previously identified in other cyanobacteria, this is the first time they have been identified from this recognized HAB species. Following their identification from C. raciborskii, the taxonomic distribution of the PMA was additionally investigated by chemical screening of a freshwater algal (i.e. cyanobacteria, green algal) culture collection. Screening suggests that these compounds are distributed among phylogenetically diverse taxa. Furthermore, parallel screening of the algal culture collection, using the zebrafish embryo model of teratogenicity, the presence of PMA was found to closely correlate with developmental toxicity of these diverse algal isolates. Taken together, the data suggest PMA contribute to the toxicity of C. raciborskii, as well as apparently several other taxonomically disparate cyanobacterial and green algal genera, and may, accordingly, contribute to the toxicity of diverse freshwater HAB.
Toxigenicity of cyanobacteria is widely associated with production of several well-described toxins that pose recognized threats to human and ecosystem health as part of both freshwater eutrophication, and episodic blooms in freshwater and coastal habitats. However, a preponderance of evidence indicates contribution of additional bioactive, and potentially toxic, metabolites. In the present study, the zebrafish (Danio rerio) embryo was used as a model of vertebrate development to identify, and subsequently isolate and characterize, teratogenic metabolites from two representative strains of C. raciborskii. Using this approach, three chemically related carotenoids - and specifically the xanthophyll glycosides, myxol 2′-glycoside (1), 4-ketomyxol 2′-glycoside (2) and 4-hydroxymyxol 2′-glycoside (3) - which are, otherwise, well known pigment molecules from cyanobacteria were isolated as potently teratogenic compounds. Carotenoids are recognized “pro-retinoids” with retinoic acid, as a metabolic product of the oxidative cleavage of carotenoids, established as both key mediator of embryo development and, consequently, a potent teratogen. Accordingly, a comparative toxicological study of chemically diverse carotenoids, as well as apocarotenoids and retinoids, was undertaken. Based on this, a working model of the developmental toxicity of carotenoids as pro-retinoids is proposed, and the teratogenicity of these widespread metabolites is discussed in relation to possible impacts on aquatic vertebrate populations.
Transmission from one host to another is a crucial component of parasite fitness. For some aquatic parasites, transmission occurs via a free‐living stage that spends time in the water, awaiting an encounter with a new host. These parasite transmission stages can be impacted by biotic and abiotic factors that influence the parasite's ability to successfully infect or grow in a new host. Here we tested whether time spent in the water column and/or exposure to common cyanobacterial toxins impacted parasite transmission stages. More specifically, we tested whether the infectivity, within host growth, and virulence of the fungal parasite Metschnikowia bicuspidata changed as a result of time spent in the water or from exposure to cyanotoxins in the water column. We exposed parasite transmission spores to different levels of one of two ecologically important cyanotoxins, microcystin‐LR and anatoxin‐a, and factorially manipulated the amount of time spores were incubated in water. We removed the toxins and used those same spores to infect one genotype of the common lake zooplankton Daphnia dentifera. We found that cyanotoxins did not impact parasite fitness (infection prevalence and spore yield per infected host) or virulence (host lifetime reproduction and survivorship) at the tested concentrations (10 and 30 μg/L). However, we found that spending longer as a transmission spore decreased a spore's chances for successful infection: spores that were only incubated for 24 hr infected approximately 75% of exposed hosts, whereas spores incubated for 10 days infected less than 50% of exposed hosts. We also found a negative relationship between the final spore yield from infected hosts and the proportion of hosts that became infected. In treatments where spores spent longer in the water column prior to encountering a host, infection prevalence was lower (indicating lower per spore infectivity), but each infected host yielded more spores at the end of infection. We hypothesise that this pattern may result from intraspecific parasite competition within the host. Overall, these results suggest that transmission spores of this parasite are not strongly influenced by cyanotoxins in the water column, but that other aspects of spending time in the water strongly influence parasite fitness.
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